Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possi...

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Authors: Guzmán Herrador, Dolores Lucía, Fernández Gómez, Andrea, Depardieu, Florence, Bikard, David, Llosa Blas, Matxalen|||0000-0002-4826-2240
Format: article
Publication Date:2024
Country:España
Institution:Universidad de Cantabria (UC)
Repository:UCrea Repositorio Abierto de la Universidad de Cantabria
Language:English
OAI Identifier:oai:repositorio.unican.es:10902/34720
Online Access:https://hdl.handle.net/10902/34720
Access Level:Open access
Keyword:CRISPR-Cas
Type IV secretion
Bacterial conjugation
Base editor
Protein translocation
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spelling Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion systemGuzmán Herrador, Dolores LucíaFernández Gómez, AndreaDepardieu, FlorenceBikard, DavidLlosa Blas, Matxalen|||0000-0002-4826-2240CRISPR-CasType IV secretionBacterial conjugationBase editorProtein translocationCRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.Acknowledgments: Work in ML lab is supported by grants PID2020-117956RB-I00 and PDC2021-120967-I00_MCIN/AEI/10.13039/501100011033_UE Next GenerationEU/PRTR from the Spanish National Research Agency (Ministry of Science and Innovation). D.B. was supported by the European Research Council [677823]; European Research Council [101044479]; and Agence Nationale de la Recherche [ANR-10-LABX-62-IBEID]. A.F.-G. was a recipient of a predoctoral appointment from the University of Cantabria. Portions of the paper were developed from the thesis of DLG-H.National Academy of SciencesUniversidad de Cantabria20242024-01-01journal articlehttp://purl.org/coar/resource_type/c_6501NAhttp://purl.org/coar/version/c_be7fb7dd8ff6fe43info:eu-repo/semantics/articlehttps://hdl.handle.net/10902/34720Proceedings of the National Academy of Sciences of the United States of America, 2024, 121(43), e2408509121reponame:UCrea Repositorio Abierto de la Universidad de Cantabriainstname:Universidad de Cantabria (UC)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:repositorio.unican.es:10902/347202026-06-02T12:39:31Z
dc.title.none.fl_str_mv Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
title Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
spellingShingle Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
Guzmán Herrador, Dolores Lucía
CRISPR-Cas
Type IV secretion
Bacterial conjugation
Base editor
Protein translocation
title_short Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
title_full Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
title_fullStr Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
title_full_unstemmed Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
title_sort Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system
dc.creator.none.fl_str_mv Guzmán Herrador, Dolores Lucía
Fernández Gómez, Andrea
Depardieu, Florence
Bikard, David
Llosa Blas, Matxalen|||0000-0002-4826-2240
author Guzmán Herrador, Dolores Lucía
author_facet Guzmán Herrador, Dolores Lucía
Fernández Gómez, Andrea
Depardieu, Florence
Bikard, David
Llosa Blas, Matxalen|||0000-0002-4826-2240
author_role author
author2 Fernández Gómez, Andrea
Depardieu, Florence
Bikard, David
Llosa Blas, Matxalen|||0000-0002-4826-2240
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidad de Cantabria
dc.subject.none.fl_str_mv CRISPR-Cas
Type IV secretion
Bacterial conjugation
Base editor
Protein translocation
topic CRISPR-Cas
Type IV secretion
Bacterial conjugation
Base editor
Protein translocation
description CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.
publishDate 2024
dc.date.none.fl_str_mv 2024
2024-01-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
NA
http://purl.org/coar/version/c_be7fb7dd8ff6fe43
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/10902/34720
url https://hdl.handle.net/10902/34720
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv National Academy of Sciences
publisher.none.fl_str_mv National Academy of Sciences
dc.source.none.fl_str_mv Proceedings of the National Academy of Sciences of the United States of America, 2024, 121(43), e2408509121
reponame:UCrea Repositorio Abierto de la Universidad de Cantabria
instname:Universidad de Cantabria (UC)
instname_str Universidad de Cantabria (UC)
reponame_str UCrea Repositorio Abierto de la Universidad de Cantabria
collection UCrea Repositorio Abierto de la Universidad de Cantabria
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869415879899348992
score 15.812429

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